Local, site-specific characteristics largely control the transmission dynamics of arthropod-borne viruses (arboviruses). Arboviruses, in turn, adapt to local conditions, maximizing their potential to perpetuate and emerge as health threats. The adaptive potential of arboviruses is driven by error-prone replication, which creates a genetically diverse pool of competing virus genotypes within each host. This application examines the influence of three important characteristics of any given transmission focus on viral genetic diversity; and how genetic diversity influences host-virus interactions. Our previous research has allowed us to make very clear predictions about each of the three examined characteristics. The frequency of contact between mosquitoes and vertebrates varies in time and space. Aim 1 will determine how the frequency of this contact influences the genetic diversity of West Nile virus (WNV) using a laboratory model transmission cycle. We predict that shorter incubation in mosquitoes allows maintenance of viral genetic diversity and high fitness. A second critical characteristic of a transmission focus is the mosquito species involved in virus transmission. Aim 2 will therefore assess how important vector mosquitoes differ in their ability to respond to WNV infection through RNA interference (RNAi) and thus drive virus diversification. Field-collected and colonized Culex (main) and Aedes (secondary) mosquitoes will be assessed. We predict that significant variation in RNAi activation will be detected within and between mosquito species, and that this variation will be associated with virus genetic diversity and altered vector competence. A third critical characteristic of a WNV transmission focus is the bird species involved in virus amplification. Aim 3, accordingly, will evaluate how crows, sparrows and robins shape WNV populations. Virus will be passed in each of these birds and genetic diversity and fitness assessed. We will also use these birds to measure the replicative fitness of different existing WNV strains and populations. We predict that wild birds exert strong purifying selection on WNV populations and that genetic diversity of the virus contributes little to its fitness in birs, but that bird infection may select for novel WNV genotypes. The significance of this work is that it will provide novel data the mechanisms that underpin the emergence of RNA viruses as health threats. The proposed research is conceptually innovative because it links the ecology of WNV transmission foci with virus adaptive plasticity. It is technically innovative because it uses uniqe methods for measuring virus fitness among relevant hosts in vivo and through its use of deep sequencing. Ultimately we seek to define the conditions that favor the emergence of novel virus genotypes. PUBLIC HEALTH RELEVANCE: This renewal aims to assess mechanisms that control WNV genetic diversity in the context of natural transmission cycles. Specifically, these studies will show how the intensity of transmission in a given focus influences the emergence of novel viral genotypes, and how important mosquitoes and birds differ in their propensity to drive virus evolution. Proposed studies are focused on WNV which is a significant and still- emerging health threat in the US, but will provide important data on how flaviviruses interact with their hosts to persist in nature and emerge as health problems.